Correlation between mechanical and enzymatic events in contracting skeletal muscle fiber

The conventional hypothesis of muscle contraction postulates that the interaction between actin and myosin involves tight coupling between the power stroke and hydrolysis of ATP. However, some in vitro experiments suggested that hydrolysis of a single molecule of ATP caused multiple mechanical cycles. To test whether the tight coupling is present in contracting muscle, we simultaneously followed mechanical and enzymatic events in a small population of cross-bridges of glycerinated rabbit psoas fibers. Such small population behaves as a single cross-bridge when muscle contraction is initiated by a sudden release of caged ATP. Mechanical events were measured by changes of orientation of probes bound to the regulatory domain of myosin. Enzymatic events were simultaneously measured from the same cross-bridge population by the release of fluorescent ADP from the active site. If the conventional view were true, ADP desorption would occur simultaneously with dissociation of cross-bridges from thin filaments and would be followed by cross-bridge rebinding to thin filaments. Such sequence of events was indeed observed in contracting muscle fibers, suggesting that mechanical and enzymatic events are tightly coupled in vivo.     

A model for the active site of skeletal muscle myosin.

A model for a main element of the active site of skeletal muscle myosin is presented that relates directly to the 92 amino acid fragment (p₁₀) of myosin recently described by Elzinga &; Collins (1977). In this model, the substrate, an eight-membered cyclic complex of MgATP, fits tightly into a 16 amino acid segment of p₁₀ and interacts with seven of its amino acids. A main feature of the model is the important role played by the one molecule of N^τ-methylhistidine² that is present in each myosin heavy chain. At the site, it is postulated that this rare amino acid functions as a donor ligand to Mg²⁺. Once N^τ-methylhistidine is put in place next to the metal, the other amino acids that appear to form a pocket come easily into position around the MgATP. These amino acids with their postulated functions are: tyrosine 72, which through a Mg-bound water, or perhaps directly, is attached to the Mg; histidine 76, which donates a proton to the P_γ of ATP; lysine 78, which binds electrostatically to P_β of ATP; phenylalanines 80 and 81, which flank the purine ring of ATP; and aspartate 66, which forms a hydrogen bond to the 6-amino group of adenine. The Mg-coordination role ascribed to N^τ-methylhistidine 69 in skeletal muscle myosin could be taken by histidine 69 in cardiac myosin and in other muscle myosins that do not contain the methylated amino acid.The choice of p₁₀ to contain a main element of the active site is based on: (a) the presence in p₁₀ of the essential sulfhydryl groups, SH1 and SH2, whose modification affects the ATPase activity of myosin; (b) the presence in ρ₁₀ of N^τ-methylhistidine, an unusual amino acid whose methylation in skeletal muscle we take as an indicator for a special function at the active site; (c) the position of p₁₀ in the primary structure near the junction between subfragment 1 and subfragment 2 (the hinge region) where, we postulate, enzymatic events at the active site are coupled to movements of the hinge that occur during contraction; (d) indications that the DTNB light chain, probably involved in regulation, is also near the hinge; (e) the effects of MgATP at the active site on the chemical reactivity of three SH groups (SH1, SH2 and SH3) located near the hinge; and (f) the effect of hinge cleavage on the oxygen exchange reaction catalyzed at the active site. The correlation of all these observations forms the basis for our placement of part of the active site on p₁₀ near the subfragment 1-subfragment 2 hinge.     

Characterization of the active site of platelet myosin in comparison to smooth and skeletal muscle myosin.

Heavy meromyosin subfragment-1 from human platelets and chicken gizzard exhibited an identical chromatographic pattern on agarose-ATP columns both in the absence and in the presence of Ca²⁺ and Mg²⁺. In the presence of Ca²⁺, the behavior differed from that of rabbit white skeletal muscle subfragment-1. The reaction of lysyl residues of platelet myosin with 2,4,6-trinitrobenzene sulfonate did not affect the K⁺- or Mg²⁺-stimulated ATPase activity. A similar behavior was exhibited by chicken gizzard myosin whereas trinitrophenylation of the more active lysyl residues in skeletal muscle myosin caused a marked increase in Mg²⁺-stimulated and a decrease in K⁺-stimulated ATPase activity. These features may point to a similar location of the essential lysyl residue in platelet and smooth muscle myosin, which is different from that of skeletal muscle. Alkylation of thiol groups by N-ethyl maleimide in the absence of added nucleotides resulted in a loss of K⁺-ATPase and in an increase in the Ca²⁺-ATPase in all three myosins, the increase for the skeletal myosin being much greater than for the platelet and chicken gizzard preparations. Alkylation of myosin in the presence of MgADP led to a decrease in K⁺-ATPase of all preparations whereas the Ca²⁺-ATPase as a function of time exhibited a maximum for the platelet and skeletal muscle proteins. These features may point to a certain similarity with respect to the active site of platelet and smooth muscle myosins and a difference between these and skeletal muscle myosin.     

Relationship between respiratory nerve and muscle activity and muscle force output.

To demonstrate the most satisfactory way of using electrical activities of respiratory nerves and muscles, activities of phrenic nerve and external intercostal muscle (ICM) and the airway pressure changes generated by respiratory muscle contraction were recorded in anesthetized cats during complete airway occlusion. Electrical activities were rectified, integrated and processed in terms of peak and average inspiratory rates per 0.1 s and of total activity per breath. Peak rate of phrenic nerve activity exhibited a high linear correlation (r = 0.974) with peak inspiratory pressure. Average phrenic rate showed a similar high correlation (r = 0.973). Peak rate of external ICM was linearly related to peak pressure but the correlation was less good (r = 0.915). Total phrenic activity per breath was too dependent upon inspiratory duration to be a satisfactory correlate (r = 0.674). In this experiment occlusion pressure was an index of muscle force generation and respiratory control system output. It is concluded that peak or average rates of phrenic activity provide an electrical index of output changes. On theoretical grounds, peak rate is probably better.     

The site of paramyosin in insect flight muscle and the presence of an unidentified protein between myosin filaments and Z-line.

The position of paramyosin in insect flight muscle was determined by labelling myofibrils with antibody to paramyosin and examining them by fluorescent and electron microscopy.Antiserum to dung beetle paramyosin had antibodies to another protein as well as to paramyosin. Specific anti-paramyosin bound to the H-zone of Lethocerus myofibrils showing paramyosin was exposed only in that region. Antibodies to the other protein bound at the ends of the A-band.The exposure of antigenic sites in the two regions of the myofibril depended on the extent of contraction in the myofibril: the sites at the end of the A-band were most exposed in rest-length myofibrils and those at the H-zone in shortened ones.Antibody-labelling in stretched bee muscle showed that the protein at the ends of the sarcomere extended from myosin filaments to Z-line.The high resting elasticity of insect flight muscle and hence its capacity for oscillatory contraction may be due to the protein between myosin filaments and Z-line.     

Cardiac myosin isoforms from different species have unique enzymatic and mechanical properties

The mammalian heart contains two cardiac myosin isoforms: beta-myosin heavy chain (MHC) is found predominantly in the ventricles of large mammals, and alpha-MHC is expressed in the atria. The sequence identity between these isoforms is approximately 93%, with nonidentical residues clustered in discrete, functionally important domains associated with actin binding and ATPase activity. It is well-established that rabbit alpha-cardiac myosin has a 2-fold greater unloaded shortening velocity than beta-cardiac myosin but a 2-fold lower average isometric force. Here, we test the generality of these relationships for another large mammal, the pig, as well as for a small rodent, the mouse, which expresses alpha-MHC in its ventricles throughout adulthood. Hydrophobic interaction chromatography (HIC) was used to purify myosin from mouse, rabbit, and pig hearts. The superior resolving power of HIC made it possible to prepare highly homogeneous, enzymatically active myosin from small amounts of tissue. The movement of actin filaments by myosin was measured in an in vitro motility assay. The same assay could be used to determine average isometric force by loading the actin filaments with increasing concentrations of alpha-actinin to stop filament motion. We conclude that myosin from the mouse has significantly higher velocities for both alpha and beta isoforms than myosin from rabbits and pigs, even though the 2-fold difference in velocity between isoforms is maintained. Unlike the larger mammals, however, the small rodent generates the same high isometric force for both alpha and beta isoforms. Thus, nature has adapted the function of cardiac myosin isoforms to optimize power output for hearts of a given species.     

Shift of binding site at the interface between actin and myosin

The molar ratio dependent change in the binding manner between actin and the lysine-rich sequence at the junction between 50K and 20K domains of subfragment 1 was studied by both protease digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The tryptic cleavage site at the function between 50K and 20K was found to be located between the third and fourth lysine residues in the lysine-rich sequence -KKGGKKK-. This site was not protected by actin when the molar ratio of actin to subfragment 1 was 1:1 but was protected at 2:1 and 3:1. The V8 protease cleavage site of chicken subfragment 1 and the elastase cleavage site of rabbit subfragment 1 were found to be located four residues away from the N-terminus of the lysine-rich sequence. Unlike the tryptic cleavage site, this site was protected by actin more when the molar ratio of actin to subfragment 1 was 1:1 than when it was 2:1 and 3:1. To understand the reason for the opposite effect of the molar ratio observed at the middle of and at four residues away from the lysine-rich sequence, actual cross-linked residue(s) was (were) determined by subjecting cross-linked product to a protein sequencer. It was found that the cross-linked sites were mainly at the first and second lysine residues of the lysine-rich sequence when the molar ratio of actin to subfragment 1 was 1:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of smooth muscle tropomyosin and smooth muscle myosin. Effect on the properties of myosin

Several techniques were used to investigate the possibility that smooth muscle tropomyosin interacts with smooth muscle myosin. These experiments were carried out in the absence of actin. The Mg2+-ATPase activity of myosin was activated by tropomyosin. This was most marked at low ionic strength but also occurred at higher ionic strength with monomeric myosin. For myosin and HMM, the activation of Mg2+-ATPase by tropomyosin was greater at low levels of phosphorylation. There was no detectable effect of tropomyosin on the Mg2+-ATPase activity of S1. The KCl dependence of myosin viscosity was influenced by tropomyosin, and in the presence of tropomyosin, the 6S to 10S transition occurred at lower KCl concentrations. From the viscosity change, an approximate stoichiometry of 1:1 tropomyosin to myosin was estimated. The phosphorylation dependence of viscosity, which reflects the 10S-6S transition, also was altered in the presence of tropomyosin. An interaction between myosin and tropomyosin was detected by fluorescence measurements using tropomyosin labeled with dansyl chloride. These results indicate that an interaction occurs between myosin and tropomyosin. In general, the interaction is favored at low ionic strength and at low levels of phosphorylation. This interaction is not expected to be competitive with the formation of the actin-tropomyosin complex, but the possibility is raised that a direct interaction between myosin and tropomyosin bound to the thin filament could modify contractile properties in smooth muscle.     

Alteration of the enzymatic properties of smooth muscle myosin by a monoclonal antibody against subfragment 2.

The fluorescent dye 10-N-nonyl acridine orange (NAO), known as specifically associated with mitochondria, has been reported to have a cytotoxic effect when high doses were applied to cells. Presently, the biochemical basis of its toxicity was investigated on isolated rat liver mitochondria. At low concentrations, NAO strongly inhibited state 3 respiration and ATP synthesis. At high concentrations, electron transport, ATP hydrolysis, P_i-transport and adenine nucleotide activities were also decreased. All these inhibitions can be explained by probe-cardiolipin interactions which could induce the collapse of energy conversion and/or the modification of membrane fluidity.     

Effects of Ca2+ on the conformation and enzymatic activity of smooth muscle myosin

The influence of Ca2+ on the enzymatic and physical properties of smooth muscle myosin was studied. The actin-activated ATPase activity of phosphorylated gizzard myosin and heavy meromyosin is higher in the presence of Ca2+ than in its absence, but this effect is found only at lower MgCl2 concentrations. As the MgCl2 concentration is increased, Ca2+ sensitivity is decreased. The concentration of Ca2+ necessary to activate ATPase activity is higher than that required to saturate calmodulin. The similarity of the pCa dependence of ATPase activity and of Ca2+ binding to myosin and the competition by Mg2+ indicate that these effects involved the Ca2+-Mg2+ binding sites of gizzard myosin. For the actin dependence of ATPase activity of phosphorylated myosin at low concentrations of MgCl2, both Vmax and Ka are influenced by Ca2+. The formation of small polymers by phosphorylated myosin in the presence of Ca2+ could account for the alteration in the affinity for actin. For the actin dependence of phosphorylated heavy meromyosin at low MgCl2 concentrations, Ca2+ induces only an increase in Vmax. To detect alterations in physical properties, two techniques were used: viscosity and limited papain hydrolysis. For dephosphorylated myosin, 6 S or 10 S, Ca2+-dependent effects are not detected using either technique. However, for phosphorylated myosin the decrease in viscosity corresponding to the 6 S to 10 S transition is shifted to lower KCl concentrations by the presence of Ca2+. In addition, a Ca2+ dependence of proteolysis rates is observed with phosphorylated myosin but only at low ionic strength, i.e. under conditions where myosin assumes the folded conformation.     

Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits

Several structural and enzymatic properties of myosin from skeletal muscles of neonatal and adult rabbits were compared. Electrophoretic analyses and proteolysis experiments indicated that differences between the two myosin types could be attributed to their heavy subunits. Circular dichroism measurements of subfragment-1 species, and trypsin-digested derivatives showed that the neonatal protein contained less alpha-helices than the adult form. The Mg2(+)-ATPase activity of neonatal myosin was lower than that of adult myosin, especially in the presence of actin. In comparison with adult subfragment-1, it was found that the binding of ATP analogues such as adenosine 5'-[beta, gamma-imino]triphosphate and PPi, or that of ATP (as deduced from the apparent KmATP) to neonatal subfragment-1 in the presence of actin was enhanced, while that of ADP was decreased. On the other hand, the association of actin with the ADP - neonatal-subfragment-1 complex was weaker. These features must be expressed in the cyclical actin-myosin association/dissociation steps occurring in ATP hydrolysis, and more particularly in the reassociation of actin with the ATP-hydrolysis-products - myosin complex.     

Interaction between troponin and myosin enhances contractile activity of myosin in cardiac muscle

Ca(2+) signaling in striated muscle cells is critically dependent upon thin filament proteins tropomyosin (Tm) and troponin (Tn) to regulate mechanical output. Using in vitro measurements of contractility, we demonstrate that even in the absence of actin and Tm, human cardiac Tn (cTn) enhances heavy meromyosin MgATPase activity by up to 2.5-fold in solution. In addition, cTn without Tm significantly increases, or superactivates sliding speed of filamentous actin (F-actin) in skeletal motility assays by at least 12%, depending upon [cTn]. cTn alone enhances skeletal heavy meromyosin's MgATPase in a concentration-dependent manner and with sub-micromolar affinity. cTn-mediated increases in myosin ATPase may be the cause of superactivation of maximum Ca(2+)-activated regulated thin filament sliding speed in motility assays relative to unregulated skeletal F-actin. To specifically relate this classical superactivation to cardiac muscle, we demonstrate the same response using motility assays where only cardiac proteins were used, where regulated cardiac thin filament sliding speeds with cardiac myosin are >50% faster than unregulated cardiac F-actin. We additionally demonstrate that the COOH-terminal mobile domain of cTnI is not required for this interaction or functional enhancement of myosin activity. Our results provide strong evidence that the interaction between cTn and myosin is responsible for enhancement of cross-bridge kinetics when myosin binds in the vicinity of Tn on thin filaments. These data imply a novel and functionally significant molecular interaction that may provide new insights into Ca(2+) activation in cardiac muscle cells.     

Acidic residues comprise part of the myosin light chain-binding site on skeletal muscle myosin light chain kinase

Myosin light chain kinase is a Ca2+/calmodulin-dependent protein kinase which exhibits a very high degree of protein substrate specificity. The regulatory light chain of myosin is the only known physiological substrate of the enzyme. Based upon epitope mapping of monoclonal antibodies which inhibit kinase activity competitively with respect to the light chain substrate, residues 235-319 of the rabbit skeletal muscle kinase have been proposed to contain a light chain-binding site (Herring, B. P., Stull, J. T., and Gallagher, P. J. (1990) J. Biol. Chem. 265, 1724-1730). With the expression of a truncated kinase, we have further localized this putative binding site to residues 235-294. Mutation of acidic residues at positions 269 and 270 of the kinase resulted in a 10-fold increase in the Km value for the myosin light chain, with no significant change in the Vmax value. In contrast, altering a cluster of acidic amino acids at positions 261-263 had little effect on the Km value for the myosin light chain. These results suggest that residues 269 and 270 may be involved in protein-substrate binding. Interestingly, these residues, located amino-terminal of the homologous catalytic core (positions 302-539), are in a region which is highly conserved among myosin light chain kinases, but not other protein kinases. It is probable that the homologous catalytic core contains structural elements required for phosphotransferase activity. The catalytic domain of myosin light chain kinase would therefore include these conserved elements together with additional specific substrate-binding residues.     

A myosin site involved in energy transduction during muscle contraction

An antibody was developed against a 23-kDa fragment of myosin which contains a part of the ATP-binding site, and applied to skinned muscle fibers. The antibody abolished active tension generation of the fibers, but did not block their assumption of a rigor state nor their release from this state by ATP. The primary amino acid sequence of the antigenic site on the fragment was found to be the region containing residues 77-80. This sequence region is predicted to have interesting secondary structures, and is distinct from the proposed ATP-binding site. We discuss the possibility that the region is responsible for the energy-transducing step in muscle contraction.     

Subunit exchange between smooth muscle myosin filaments

Filaments formed from phosphorylated smooth muscle myosin are stable in the presence of MgATP, whereas dephosphorylated filaments are disassembled to a mixture of folded monomers and dimers. The stability of copolymers of phosphorylated and dephosphorylated myosin was, however, unknown. Gel filtration, sedimentation velocity, and pelleting assays were used to show that MgATP could dissociate dephosphorylated myosin from copolymers containing either rod and myosin or dephosphorylated and phosphorylated myosin. Copolymers were typically formed by dialyzing monomeric mixtures into filament-forming buffer but, unexpectedly, could also be formed within minutes of mixing preformed rod and myosin minifilaments. This result suggested that molecules can rapidly and extensively exchange between filaments, presumably via the monomeric pool of myosin in equilibrium with polymer. An exchange of molecules between filaments was demonstrated directly by electron microscopy using gold-labeled streptavidin or antibody to detect the exchanged species. By this approach it was shown that smooth muscle myosin filaments, like other macromolecular assemblies, are dynamic structures that can readily alter their composition in response to changing solvent conditions. Moreover, because folded monomeric myosin is unable to polymerize, these experiments suggest a mechanism for the disassembly of the filament by MgATP.     

Active site comparisons highlight structural similarities between myosin and other P-loop proteins.

The phosphate binding loop (P-loop) is a common feature of a large number of enzymes that bind nucleotide whose consensus sequence is often used as a fingerprint for identifying new members of this group. We review here the binding sites of nine purine nucleotide binding proteins, with a focus on their relationship to the active site of myosin. This demonstrates that there is considerable conversation in the distribution and nature of the ligands that coordinate the triphosphate moiety. This comparison further suggests that at least myosin and the G-proteins utilize a similar mechanism for nucleotide hydrolysis.